© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2996-3008 doi:10.1242/jcs.163964

RESEARCH ARTICLE A role for Rab23 in the trafficking of Kif17 to the primary cilium Yi Shan Lim1 and Bor Luen Tang1,2,*

ABSTRACT midbrain and hindbrain regions, and do not survive beyond the The small GTPase Rab23 is an antagonist of (Shh) second half of gestation (Eggenschwiler et al., 2001; Gunther et al., signaling during mouse development. Given that modulation of Shh 1994). How exactly Rab23 silences the Shh pathway in dorsal neural signaling depends on the normal functioning of the primary cilium, cells and how Rab23 controls Shh target , is unknown. In light and overexpression of Evi5L, a putative Rab23 GTPase-activating of the general function of Rabs, Rab23 could potentially regulate (GAP), leads to reduced ciliogenesis, Rab23 could have a role trafficking of Shh signaling components (Lim et al., 2011; Wang at the primary cilium. Here, we found that wild-type Rab23 and the et al., 2006), but evidence for this is still lacking. Rab23 has been constitutively active Rab23 Q68L mutant were enriched at the primary shown to work distally and downstream of both patched and cilium. Therefore, we tested the role of Rab23 in the ciliary targeting of smoothened, but upstream of the Gli transcription factors known cargoes and found that ciliary localization of the -2 (Eggenschwiler et al., 2006; Evans et al., 2003). motor protein Kif17 was disrupted in Rab23-depleted cells. The widespread distribution of Rab23 across metazoans Co-immunoprecipitation and affinity-binding studies revealed that suggests that there is a fundamental physiological role of Rab23 Rab23 exists in a complex with Kif17 and importin β2 (the putative beyond Shh signaling (Guo et al., 2006; Lumb and Field, 2011). Kif17 ciliary import carrier), implying that Kif17 needs to bind to In addition, high Rab23 expression levels in adult mouse brains regulatory like Rab23 for its ciliary transport. Although a hinted at a postnatal function beyond embryogenesis. Elevated ciliary–cytoplasmic gradient of nuclear is necessary to regulate Rab23 has been found in hepatocellular carcinomas (HCC) (Liu the ciliary transport of Kif17, Rab23 and Ran appear to have differing et al., 2007; Sun et al., 2012), lung cancer tissues (Huang et al., roles in regulating the ciliary entry of Kif17. Our findings have 2011) and atrophic gastritis with intestinal metaplasia (Kim et al., uncovered a hitherto unknown effector of Rab23 and demonstrate 2007), and it is also associated with diffuse-type gastric cancer how Rab23 could mediate the transport of Kif17 to the primary cilium. (dGC) (Hou et al., 2008) and the follicular adenoma type of thyroid tumors (Denning et al., 2007). Besides tumorigenesis, KEY WORDS: Rab23, Kif17, Importin β2, Primary cilium, Trafficking Rab23 is also upregulated in kidney mesangial cells of focal segmental glomerulosclerosis (FSGS) mice (Huang et al., 2009; INTRODUCTION Shui et al., 2008). In humans, homozygous nonsense mutations in Rab23 belongs to the subfamily, the largest branch of the Ras Rab23 result in a congenital pleiotrophic disorder – Carpenter’s superfamily of small . To date, more than 60 Rabs and Rab- syndrome – where patients suffer from anatomical and like proteins have been identified (Elias et al., 2012; Grosshans physiological deformities (Alessandri et al., 2010; Jenkins et al., et al., 2006; Pfeffer, 2005) but the functions of only about 36 Rabs 2007). Although these Rab23-associated diseases could be linked are known (Schwartz et al., 2007). Rab GTPases are known to to dysregulated Shh signaling in postnatal life (Ruiz i Altaba et al., localize to distinct membrane-bound compartments, conferring 2002), Rab23 could also have physiological functions beyond vesicle and organelle membrane identities (Sonnichsen et al., 2000). embryogenesis and Shh signaling (Chia and Tang, 2009). More importantly, they orchestrate multiple steps of vesicular Rab23 is a candidate ciliary protein because it has been identified transport between different organelles of the endocytic and in the ciliary proteome of isolated mouse kidney primary cilia secretory pathways in a spatial and temporal manner (Schwartz (Ishikawa et al., 2012) and the Ciliary Proteome Database version 3 et al., 2007; Zerial and McBride, 2001). (Gherman et al., 2006). It is also present at the flagellum of The first insight into the function of Rab23 came from cloning of Trypanosoma brucei throughout its life cycle (Lumb and Field, the mouse open brain (opb) allele (Eggenschwiler and Anderson, 2011). In fact some features of the human Carpenter’s syndrome, for 2000; Gunther et al., 1994; Sporle et al., 1996) of Rab23 instance cranial malformations, obesity and polysyndactyly, do (Eggenschwiler et al., 2001). In vertebrate embryonic patterning, resemble those frequently observed in (Mykytyn et al., Rab23 is required for dorsalization and acts antagonistically to Shh, 2002). Intriguingly, studies have shown that overexpression of which is essential for ventral cell type specification (Chiang et al., Evi5L, a Rab23 cognate GTPase-activating protein (GAP), leads to 1996; Sporle et al., 1996). Successful sequential activation of both reductions in ciliation (Yoshimura et al., 2007), and that Rab23 genes is crucial in Shh-dependent patterning of the neural tube, and modulates recycling of the Shh signaling activator smoothened at normal development of the brain and spinal cord. Rab23 mutant the primary cilium (Boehlke et al., 2010). Although this implies that mouse embryos exhibit exencephalic malformation of the forebrain, Rab23 is associated with the primary cilium, the exact function of Rab23 and its exact mode of action remain obscure. We report here a previously unknown cilia-associated function of 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, 8 Medical Drive, 117597 Singapore. 2National University of Rab23 and that Rab23 could be regulating the ciliary transport of Singapore Graduate School of Integrative Sciences and Engineering, National Kif17. Our results suggest that one role of Rab23 could be to University of Singapore, 28 Medical Drive, 117456 Singapore. facilitate binding of Kif17 with its cognate ciliary import carrier β *Author for correspondence ([email protected]) importin 2 [also known as transportin 1 (TNPO1) or karyopherin β2]. Formation of such a tripartite complex is likely to be essential

Received 29 September 2014; Accepted 25 June 2015 for targeting Kif17 to the primary cilium. Journal of Cell Science

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RESULTS Rab23 has a role in the ciliary trafficking of Kif17 Wild-type Rab23 and Rab23 Q86L localize to the cilia We next asked whether Rab23 could be involved in the Despite several investigations into functional associations between trafficking of known ciliary proteins, such as the Shh signaling Rab23 and the primary cilium, it remains uncertain whether Rab23 is transducer smoothened (Wang et al., 2009), Arl13b, a ciliary indeed present at the primary cilium. Immunostaining with anti-Rab23 membrane-associated GTPase mutated in antibodies (Guo et al., 2006) has shown that endogenous Rab23 is (Cantagrel et al., 2008), and Kif17, a soluble kinesin-2 family generally cytosolic and associated with subcellular membranous motor protein responsible for transport processes along axonemal structures, but that its localization at the primary cilia cannot be easily microtubules (Hirokawa et al., 2009; Verhey et al., 2011). discerned. To test whether overexpression of Rab23 could demonstrate Although there were no significant differences observed in the clearer cilia localization, cells expressing wild-type Rab23, GTPase- ciliary localization of Arl13b after Rab23 silencing in NIH3T3 activity-deficient Rab23 Q68L (constitutively active), GTP-binding- fibroblasts (supplementary material Fig. S1D–F), Rab23-silenced defective Rab23 S23N (dominant negative) and the non- cells exhibited a moderate increase in the intensity of ciliary phosphorylatable Rab23 T150A mutants (hereafter denoted SmoothenedA1 (SmoothenedA1 is a constitutively active W549L Rab23WT, Rab23QL, Rab23SN and Rab23TA, respectively) were mutant) (supplementary material Fig. S1A,B). This corroborated compared. Overexpressed mCherry-tagged Rab23WT and Rab23QL the previously defined role of Rab23 as a negative regulator of displayed distinct colocalization with the primary cilia marker the Shh pathway (Eggenschwiler et al., 2006) and the finding acetylated α- in NIH3T3 (Fig. 1A), human (h)TERT- that Rab23 regulates the recycling of ciliary smoothened immortalized retinal pigment epithelial (hTERT-RPE1) (Boehlke et al., 2010), explaining how loss of Rab23 could (supplementary material Fig. S2A) and CRL1927 mouse mesangial lead to an accumulation of SmoothenedA1 at the primary cilium. cells (data not shown). Although a majority of mCherry–Rab23WT However, in our hands, immunoprecipitation of Rab23 with anti- and –Rab23QL cells localized to the primary cilia of NIH3T3 cells, Rab23 antibodies did not show an interaction with only ∼18% of Rab23TA cells exhibited Rab23 ciliary localization, SmoothenedA1 (data not shown). At this point, it is unclear whereas overexpressed Rab23SN could not be detected at the cilia how exactly Rab23 could be involved in the ciliary trafficking of (Fig. 1B). This differential enrichment in the cilia was observed smoothened or in the modulation of Shh signaling. despite comparable expression levels of Rab23 constructs after In investigating Kif17 as a potential ciliary cargo of Rab23, transient transfection in NIH3T3 cells (Fig. 1C). Similarly, however, we found that the ciliary tip localization of Kif17 was overexpressed untagged Rab23WT and Rab23QL mutant, when significantly enhanced after exogenous expression of mCherry– immunostained with anti-Rab23 antibodies, were also enriched at the Rab23WT and –Rab23QL. This was in contrast to a drastic primary cilia (Fig. 1D). These results suggest that the degree of Rab23 reduction in Kif17 signals in Rab23SN-expressing cells and a ciliary localization is dependent on the GTP-binding status of Rab23. more moderate reduction in the phosphorylation mutant In addition, disruption of phosphorylation site at T150 of Rab23 also Rab23TA-expressing cells (Fig. 2A,B). For the populations appeared to affect the targeting of Rab23 to the primary cilium. expressing Rab23SN and Rab23TA mutant proteins, there were Before cell fixation, a brief incubation with 0.1% Triton X-100 also significantly fewer Kif17-positive cilia (Fig. 2C). To can be used to extract ciliary membrane proteins and release other overcome any possible effects of Rab23 manipulation on membrane and cilioplasmic proteins, but leave the ciliary axoneme ciliogenesis that might indirectly impinge on ciliary transport, intact. Incubation with Triton X-100 resulted in a loss of mCherry– cells were serum-starved to induce cilium formation before Rab23QL staining along the primary cilium, whereas acetylated α- carrying out the Rab23-knockdown treatments. Similar to what tubulin staining of the axoneme remained (Fig. 1E, top two panels), was observed after mCherry–Rab23SN overexpression, Rab23 suggesting that Rab23 is not tightly associated with the ciliary silencing resulted in significant decreases in the ciliary intensities axoneme but is more likely to be mostly bound to the ciliary of Kif17 and the Kif17-positive cilia number in NIH3T3 membrane or existing in a soluble form within the cilioplasm. This fibroblasts (Fig. 2D–F). As overexpression of dominant- loss was also observed with a known ciliary membrane protein, negative Rab23SN in NIH3T3 cells led to stunted cilial growth ADP-ribosylation factor-like 13b (Arl13b) (Fig. 1E, lower two that could adversely affect ciliary transport, the phenotype panels) (Larkins et al., 2011). elicited by Rab23SN overexpression should be interpreted with The ciliary enrichment of GTP-Rab23 hinted at a possible role caution. The Kif17 phenotype elicited by the Rab23 dominant- for Rab23 in the morphological development of primary cilia. We negative mutant is nonetheless similar to the Rab23-silencing therefore checked the effect of Rab23 manipulation on ciliogenesis phenotype. To further validate that perturbation of the in NIH3T3 fibroblasts. Compared to mCherry-tagged Rab23WT, localization of Kif17 was more likely due to defective ciliary Rab23QL, Rab23TA and mock-transfected (‘MT’)control entry of Kif17 rather than an inhibition of Kif17 transcription, we NIH3T3 cells, only Rab23SN-expressing cells exhibited a showed that Kif17–mCitrine expression levels were relatively significant decrease in ciliation and shorter cilia lengths. By unaffected after Rab23 silencing (Fig. 2G). contrast, mCherry–Rab23WT and –Rab23QL cells grew When the Rab23 manipulations were repeated in hTERT-RPE1 moderately longer cilia than vector control cells (Fig. 1F). epithelial cells, the ciliary intensities of Kif17 were also enhanced in Although the results imply that higher levels of ciliary GTP– cells overexpressing Rab23WT and Rab23QL, and significantly Rab23 present can be correlated with increased ciliary growth, reduced in cells overexpressing Rab23SN compared to vector Rab23 silencing did not seem to have an overall adverse effect on control (supplementary material Fig. S2A,B). This was observed cilia morphology in NIH3T3 fibroblasts (Fig. 1G,H). Expression despite no significant differences in Kif17-positive cilia numbers of the dominant-negative Rab23SN mutant thus possibly had among the differently transfected cells (supplementary material Fig. indirect or non-Rab23-specific consequences, which led to the S2C). By contrast, the ciliary localization of Kif17 was significantly markedly more severe ciliogenesis phenotype. Based on these disrupted in serum-starved Rab23-silenced cells (supplementary results, however, loss of Rab23 does not appear to have a crucial material Fig. S2D–F), and this was again observed despite effect on ciliogenesis in NIH3T3 fibroblasts. comparable expression levels of Kif17–mCitrine between Journal of Cell Science

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Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. Rab23 localizes to the primary cilia of NIH3T3 fibroblasts but might Rab8a has been previously shown to have a role in the ciliary not have a crucial role in cilium biogenesis. (A) Cells expressing mCherry- transport of a C-terminal fragment of fibrocystin (harboring the tagged Rab23WT, Rab23QL, Rab23SN and Rab23TA, and vector control (red) ciliary-targeting sequence, CTS) in mouse kidney inner medullary were induced to form primary cilia by serum starvation, and later incubated with antibodies against the primary cilia marker acetylated (Ac.) α-tubulin (green) collecting duct (IMCD3) cells (Follit et al., 2010). In agreement and with Hoechst 33342 (blue) for the staining of nuclei. Colocalization of with these results, Rab8a knockdown resulted in a significant loss of Rab23 and acetylated α-tubulin is observed in both x-y planes and z-views (of ciliary fibrocystin-CTS (supplementary material Fig. S4A–D) in different cells). (B) Quantification of the percentage of ciliated cells exhibiting NIH3T3 cells. In contrast, Rab23 was not important in modulating Rab23 ciliary localization following transfection of the various mCherry–Rab23 ciliary transport of fibrocystin-CTS, as Rab23 silencing did not n≥ constructs ( 60 per condition; pairwise comparisons made between affect the ciliary localization of fibrocystin-CTS (supplementary mCherry–Rab23WT and Rab23TA or Rab23QL cells; ***P<0.001). – (C) Representative western blot (WB) showing expression levels of mCherry– material Fig. S4F I). Rab23 constructs after transient transfection in NIH3T3 fibroblasts. β-actin was To better examine how Rab23 could be influencing the ciliary probed as a loading control and approximate molecular sizes in kDa are shown transport kinetics of ciliary Kif17, we used fluorescence recovery on the left of blot. (D) Untagged Rab23WT, Rab23QL, Rab23SN and Rab23TA after photobleaching (FRAP). After laser-pulse-induced exhibited a similar ciliary localization pattern to their mCherry–Rab23 photobleaching of the Kif17 signal at the ciliary tip, we compared counterparts. Cells were fixed and stained with anti-Rab23 (red) and the recovery of the normalized, mean Kif17 fluorescence intensity at α anti-acetylated -tubulin (green) antibodies, and Hoechst 33342 (blue). the bleached region between scrambled and Rab23-specific (E) Comparison of ciliary staining of Rab23QL and Arl13b with and without Triton X-100 treatment prior to cell fixation. First and second rows, cells shRNA-treated cells. Whereas scrambled cells could recover up to expressing mCherry–Rab23QL (red) were immunolabeled for acetylated ∼30% of initial ciliary Kif17 intensity, Rab23-silenced cells α-tubulin (green) and pericentrin (purple); third and fourth rows, cells exhibited little or no recovery of the ciliary Kif17 signal over time transfected with Arl13b–GFP (green) were stained for acetylated α-tubulin (Fig. 4). Collectively, the results so far strongly indicate that Rab23 (red) and pericentrin (purple). Nuclei of these cells were counterstained with is important for the entry of Kif17 into primary cilia. Hoechst 33342 dye (blue). For A, D and E, white boxes highlight cilia locations. μ Scale bars: 5 m. (F) Left chart, compared to mock-transfected (MT) cells, β vector control cells, and cells expressing mCherry-tagged Rab23WT, Rab23 interacts with Kif17 and importin 2 Rab23QL and Rab23TA, Rab23SN-expressing cells exhibited a significant Kif17 has been shown to depend on nuclear transport adaptor decrease in ciliation (n>100 for each condition; pairwise comparisons were importin β2 for its import into the primary cilium (Dishinger et al., made between vector control and cells overexpressing mCherry-tagged 2010). To test how Rab23 could be involved with its potential Rab23WT, Rab23QL, Rab23SN or Rab23TA; ***P<0.001). Right chart, ciliary cargo Kif17 and its putative transport adaptor importin β2, – mCherry Rab23QL cells exhibited significantly longer ciliary lengths, whereas co-immunoprecipitation analysis was performed to check for mCherry–Rab23SN cells had shorter cilia compared to vector control cells (n>30 for each condition; pairwise comparisons were made between vector possible interaction between the three proteins. Using anti-Rab23 control and cells overexpressing mCherry-tagged Rab23WT, Rab23QL, antibodies for immunoprecipitation experiments in lysate from Rab23SN or Rab23TA; ***P<0.001). (G) Left chart, the percentages of ciliated Kif17–mCitrine- and Rab23WT-transfected cells, Rab23 was found cells were tabulated for scrambled control and Rab23-siRNA-treated cells to co-precipitate with endogenous importin β2 and Kif17–mCitrine. (siRNA7.2 and siRNA3) (n>300 for each condition; pairwise comparisons were Both interactions were enhanced upon addition of the non- made between scrambled control and siRNA7.2- or siRNA3-treated cells; hydrolyzable GTP analog GTPγs, with respect to untreated cell P ** <0.01). Right chart, cilia lengths do not differ between scrambled control lysate, as well as the negative controls (cell lysate treated with GDP and Rab23-knockdown cells (n>60 for each condition; pairwise comparisons were made between scrambled control and siRNA7.2- or siRNA3-treated and cell lysate incubated with rabbit control IgG) (Fig. 5A). In the cells). (H) Quantification of efficiency of Rab23 silencing by siRNA7.2 and reciprocal co-immunoprecipitation using anti-importin β2 siRNA3 sequences. Densitometric analysis was performed based on western antibodies, Rab23 binding to Kif17–mCitrine and importin β2 blots from three independent experiments (***P<0.001). also increased in a GTP-dependent manner (Fig. 5B). This interaction between Rab23, Kif17–mCitrine and importin β2 scrambled control and Rab23-knockdown cells (supplementary could also be observed in hTERT-RPE1 cells (supplementary material Fig. S2G). material Fig. S2H). Notably, Rab8a, whose manipulation did not To show that ciliary mislocalization of Kif17 was due to a perturb Kif17 ciliary localization, did not interact with either Kif17 specific effect of Rab23 depletion, the presence of Kif17 at the or importin β2 (supplementary material Fig. S3F). By contrast, primary cilia was assessed after expression of a non-degradable whereas Rab8a interacted with fibrocystin-CTS (supplementary form of Rab23WT in Rab23 short hairpin RNA (shRNA)-silenced material Fig. S4E), there was no detectable interaction between cells. Transfection of mCherry–Rab23WT in Rab23-shRNA- Rab23 and fibrocystin-CTS (supplementary material Fig. S4J). treated cells led to increases in the ciliary localization of Kif17 One plausible reason for Rab23 existing in a complex with compared to Rab23-depleted and even scrambled control cells, with importin β2 and Kif17 would be that it facilitates the binding of significantly higher Kif17 ciliary intensities and numbers of Kif17- Kif17 to importin β2. The affinity of importin β2 for Kif17 was positive cilia (Fig. 3). hence assessed after Rab23 knockdown. Whereas Kif17–mCitrine Interestingly and importantly, we found that the ciliary and importin β2 binding increased in the presence of GTPγsin localization of Kif17 was rather specifically dependent on Rab23, scrambled cell lysate, there was no detectable interaction between but not another known ciliary Rab GTPase, Rab8a, which has been importin β2 and Kif17 in Rab23 knockdown cell lysate, even in the previously established as being essential for cilium formation and presence of GTPγs (Fig. 5C). Sufficient levels of Rab23 could ciliary cargo transport (Yoshimura et al., 2007; Nachury et al., 2007; therefore be required for binding of Kif17 to importin β2, and this Follit et al., 2010). Unlike in Rab23SN-expressing cells, dominant- requirement could underlie the Rab23-mediated regulation of Kif17 negative Rab8aTN did not diminish the localization of Kif17 at the ciliary transport. ciliary tip (supplementary material Fig. S3A). Likewise, depletion To investigate whether Rab23 interacts directly with Kif17 of endogenous Rab8a, unlike after Rab23 knockdown (Fig. 2D–G), in vitro, we produced recombinant glutathione-S-transferase did not result in a significant disruption of the ciliary localization of tagged wild-type Rab23 (GST–Rab23) and hexahistidine-

Kif17 (supplementary material Fig. S3B–E). tagged Kif17 (His6–Kif17) in bacteria. GST–Rab23-bound Journal of Cell Science

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Fig. 2. Activity of Rab23 and its expression is required for ciliary localization of Kif17. (A) NIH3T3 fibroblasts were co-transfected with a Kif17–mCitrine- bearing construct (green), and mCherry empty vector or constructs bearing mCherry-tagged Rab23WT, Rab23QL, Rab23SN or Rab23TA (red) before incubation with antibody against acetylated (Ac) α-tubulin (purple) and Hoechst 33342 dye (blue). (B) The ciliary localization of Kif17 was quantified by measurement of the intensities of Kif17 at distal ciliary ends of overexpressing cells (n≥60 per condition; pairwise comparisons made between vector control and mCherry-Rab23WT, Rab23QL, Rab23SN or Rab23TA expressing cells; *P<0.05 and ***P<0.001). (C) The number of Kif17-positive cilia among various transfected cells was also scored (pairwise comparisons made between vector control cells and cells overexpressing mCherry-tagged Rab23WT, Rab23QL, Rab23SN or Rab23TA; *P<0.05 and **P<0.01). (D) Ciliary localization of Kif17 was assessed in serum-starved NIH3T3 cells after treatment with scrambled control siRNA and mouse Rab23-specific siRNA7.2 and siRNA3. Fixed cells were stained for acetylated α-tubulin (red), pericentrin (purple) and with Hoechst 33342 (blue). (E) The ciliary intensities of Kif17 were measured in scrambled control and Rab23-depleted cells (n>60 per condition; pairwise comparisons made between scrambled control, siRNA7.2- or siRNA3-treated cells; ***P<0.001). (F) In addition to counting of Kif17-localized cilia, Kif17-positive cilia were further categorized based on its localization at the distal ciliary tips. Marking of basal bodies with pericentrin helped indicate the orientation of the primary cilium (pairwise comparisons made between scrambled control, and siRNA7.2- or siRNA3-treated cells; ***P<0.001). (G) Top panels, representative western blot showing Rab23 and Kif17–mCitrine expression levels in serum-starved NIH3T3 cells transfected with scrambled control (Scr) and Rab23 siRNA7.2 (si7.2) or siRNA3 (si3). The bottom graph shows a densitometric analysis of Rab23 and Kif17–mCitrine protein levels consolidated from three independent experiments (***P<0.001). β-actin is probed as a loading control. For A and D, white boxes highlight cilia locations. Results in B and E are shown as box plots, as described in the Materials and Methods section. Scale μ bars: 10 m. Journal of Cell Science

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Fig. 3. Expression of non-degradable Rab23 restores the ciliary localization of Kif17 in Rab23-knockdown cells. (A) The loss of ciliary localization of Kif17 in Rab23-silenced NIH3T3 cells was rescued by expression of non-degradable mCherry–Rab23WT (mC-Rab23WT). Notably, Rab23 was depleted by retroviral transduction of shRNA targeting the 5′ UTR of mouse Rab23. shRNA-treated cells transfected with mCherry–Rab23WT (red) and/or Kif17–mCitrine (green) were incubated with anti-acetylated α-tubulin (red in first two rows; purple in third row) and anti-pericentrin (purple in first two rows) antibodies, and with Hoechst 33342 (blue). White-boxed regions and arrows highlight locations of representative cilia that are further magnified (right). Scale bars: 10 μm. (B,C) Quantification of the ciliary localization of Kif17 showed both its ciliary intensities and the number of Kif17-positive cilia number significantly increased in Rab23-silenced cells rescued with mCherry–Rab23WT (n=60 per condition; pairwise comparisons made between scrambled control and Rab23-shRNA-treated or mCherry– Rab23WT-rescued cells; **P<0.01 and ***P<0.001). Results in B are shown as a box plot, as described in the Materials and Methods section. glutathione beads were used in an affinity binding (pulldown) from the cell lysate. Interestingly, but not unexpectedly, a analysis. However, GST–Rab23 could not pull down purified fraction of Kif17 was co-depleted with importin β2. GST–Rab23 His6–Kif17 (data not shown). Although in vitro binding studies could still pull down an appreciable quantity of Kif17 from the appear to indicate a lack of direct interaction between both importin-β2-depleted lysate (Fig. 5E). We quantified the proteins, this was not unexpected as Rab23 possibly requires respective signal ratio of Kif17 and importin β2intheGST– activation by a yet unknown guanine exchange factor Rab23 pulldown versus that of the lysate (input) (Fig. 5E), and it (GEF) not present in bacteria. Indeed, GST–Rab23 was able to appeared that GST–Rab23 pulled down more Kif17 than pull down both endogenous Kif17 and importin β2 in cell lysate importin β2 relative to their respective amounts in the cell in a GTPγS-enhanced manner (Fig. 5D). We attempted to lysate. The experiments described above suggest that Rab23 investigate whether importin β2 is important for the affinity of interacts with Kif17 and importin β2 in a GTP-dependent manner

GST–Rab23bindingofKif17byimmuno-depletingimportinβ2 in cell lysates. However, the difference in the antibodies used and Journal of Cell Science

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Fig. 4. Loss of endogenous Rab23 results in significantly slower recovery of ciliary Kif17 after photobleaching. (A) FRAP analysis of NIH3T3 cells expressing Kif17–mCherry (red) incubated with retrovirus harboring either scrambled control or Rab23- specific shRNA sequences (in pUEG-GFP vector; green). Representative images were captured at specific time-points: pre-bleach (after 2 min), post-bleach (after 3 min) and recovery (20 min). White arrowheads indicate representative cilia, and white boxed insets are a 10× magnification of the respective regions. Scale bars: 10 μm. (B) Ciliary fluorescence of Kif17 recovery in both scrambled control and Rab23-knockdown cells was quantified over a time span of 20 min. Data is presented as mean±s.e.m.; n=9 per condition. (C) Top panels, representative western blot of Rab23 expression in cells treated with scrambled control (Scr) and Rab23 shRNA. Bottom graph, Rab23 protein levels were consolidated from three independent experiments (***P<0.001) and normalized against β-actin (loading control).

lack of a way to accurately determine the respective molar ratios different modes of ensuring that Kif17 is imported into the ciliary of Kif17 and importin β2capturedbyGST–Rab23 did not permit compartment. Hence, although activated Ran promotes dissociation any conclusive inference on direct binding between these of importin β2 from Kif17, activated Rab23 enhances their molecules. association.

Comparing the roles of Ran and Rab23 roles in regulating DISCUSSION ciliary import of Kif17 Localization of Rab23 at the primary cilium Together with the potential role of importin β2 in the transport of There is controversy regarding the localization of Rab23 at the ciliary Kif17, Verhey’s group also discovered that maintenance of primary cilium. Although Yoshimura and co-authors did not an asymmetric distribution of Ran–GTP across the cytoplasm and observe Rab23 ciliary localization after overexpression of GFP– cilium is essential for the ciliary entry of Kif17 (Dishinger et al., Rab23WT in hTERT-RPE1 cells (Yoshimura et al., 2007), 2010). Taking the current findings into consideration, there are now overexpressed Rab23 has been reported to localize to the cilia of two small GTPases influencing ciliary transport of Kif17. It is Madin–Darby canine kidney (MDCK) cells (Boehlke et al., 2010). unclear how the influence of Rab23 on Kif17 ciliary trafficking In our hands, endogenous Rab23 was not noticeable at the primary could be functionally connected to the Ran-mediated mechanism. cilia, but overexpressed wild-type Rab23 and Rab23QL displayed We also observed that abolishment of the ciliary–cytoplasmic Ran- distinct ciliary localization in NIH3T3 (Fig. 1), hTERT-RPE1 cells GTP–Ran-GDP gradient across the ciliary base (by overexpressing (supplementary material Fig. S2A) and CRL1927 mouse mesangial constitutively active mCherry–RanQ69L) led to a significant cells (data not shown). By contrast, dominant-negative Rab23SN perturbation of the ciliary localization of Kif17 in NIH3T3 was not present at the primary cilia in these cells, whereas the fibroblasts (data not shown). To ask whether Rab23 and Ran phosphorylation mutant Rab23TA exhibited reduced Rab23 ciliary could be working cooperatively or in distinct pathways, ciliary localization. Given that ciliary enrichment of Rab23 appeared to be Kif17 was quantified after combined silencing of Rab23 and mostly associated with GTP-bound forms, the ciliary targeting of overexpression of either mCherry–RanQ69L or dominant negative Rab23 could well depend on its GTP-binding ability. RanT24N mutant. Intriguingly, siRNA-mediated depletion of In a mass spectrometric analysis, Rab23 was identified as a Rab23 in cells expressing RanQ69L exhibited the largest protein phosphorylated by cytidine 3′,5′-cyclic monophosphate reduction in ciliary intensities of Kif17 (Fig. 6A,B). This additive (cCMP) (Bond et al., 2007). Following this finding, no functional loss of ciliary Kif17 suggests that Rab23 or Ran could have significance had been associated with this phosphorylation. independent roles in regulating Kif17 transport to the primary Examining the ciliary localization of Rab23WT and Rab23TA has cilium. Notably, overexpression of Rab23QL, but not RanQ69L, led us to discover that the T150 phosphorylation could serve to resulted in a detectable interaction between importin β2 and Kif17– regulate ciliary localization of Rab23 (Fig. 1A,B,D). Pre-fixation mCitrine (Fig. 6D), further implying that Rab23 and Ran have detergent treatment of Rab23QL-expressing cells (Fig. 1E) further Journal of Cell Science

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Fig. 5. Rab23 exists in a complex with Kif17 and importin β2. (A) NIH3T3 cells co-expressing Rab23WT and Kif17–mCitrine were lysed and were either left untreated (–, lane 2), or were treated with GTPγS (lane 3) or GDP (lane 4). Co-immunoprecipitation (IP) was carried out using rabbit polyclonal anti-Rab23 antibodies, with rabbit control IgG antibodies included as a negative control (IgG, lane 5). To show loading levels of Rab23 immunoprecipitates, Rab23 levels were probed (IB) with anti-Rab23 antibody and the presence of Kif17–mCitrine and importin β2 was detected (WB) using anti-GFP and anti-transportin antibodies, respectively. 20 μg of total lysate was loaded as input (lane 1). (B) In the reciprocal co-immunoprecipitation, anti-transportin antibodies were used for immunoprecipitation and immunoprecipitates were probed for Kif17–mCitrine and Rab23 using anti-GFP and anti-Rab23 antibodies, respectively. An immunoblot of importin β2 protein levels (lowest panel) indicates similar loading amounts. 15 μg of total lysate was loaded as input. (C) Scrambled control (sc) or Rab23- specific siRNA7.2 (si)-treated cells were additionally transfected with Kif17–mCitrine. Harvested cell lysates were then either left untreated (–) or treated with GTPγs before immunoprecipitation was performed with anti-transportin antibodies. The presence of Kif17–mCitrine was detected using anti-GFP antibody, whereas importin β2 was probed with anti-transportin antibody. 20 μg of total lysate (per condition) were loaded as inputs (lanes 1 and 2). (D) GST- and GST– Rab23WT-bound glutathione beads were incubated with NIH3T3 cell lysate that was left untreated (–), or was treated with GTPγs or GDP. The presence of Kif17 and importin β2, were probed (WB) using anti-Kif17 and anti-transportin antibodies. 30 μg of total lysate was loaded as input and a Coomassie-Blue-stained gel (shown in the bottom panel) indicates the levels of GST and GST–Rab23WT proteins used in the affinity pull down. (E) Representative blot showing the levels of Kif17 [detected with anti-Kif17 (WB)] and importin β2 (probed using anti-transportin) were reduced in NIH3T3 cell lysate depleted of importin β2 (pre-cleared using mouse polyclonal anti-transportin; Pre-cleared) as compared to mock-depleted lysate (incubated with mouse IgG; Mock) in the presence of GTPγS. 30 μg of total lysate was loaded as input and a Coomassie-Blue-stained gel is included to indicate similar levels of GST and GST–Rab23WT proteins used during affinity pull down. Bottom graph, signal ratio of GST–Rab23WT pull down versus input were determined by densitometry analyses of three independent blots and tabulated as mean±s.e.m. for Kif17 and importin β2, respectively (***P<0.001). For all panels, molecular sizes in kDa are shown on the left of blots. All blots displayed are representative of at least two independent experiments. Journal of Cell Science

3003 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 2996-3008 doi:10.1242/jcs.163964

Fig. 6. Rab23 and Ran function independently in regulating the ciliary localization of Kif17. (A) NIH3T3 fibroblasts were first incubated with either scrambled control siRNA (scRNA) or Rab23-specific siRNA7.2 before co-transfection with Kif17–mCitrine (green) and mCherry-tagged RanQL, RanTN or vector control (red). Fixed cells were stained for primary cilia and nuclei using anti-acetylated α-tubulin antibody (purple) and Hoechst 33342 (blue). White boxed regions indicate representative cilia. Scale bars: 10 μm. (B) Compared to other transfected cells, Rab23-silenced cells expressing mCherry–RanQL exhibited the most significant reduction in the ciliary intensity of Kif17 (n≥60 per condition; pairwise comparisons made between vector control and cells overexpressing mCherry-tagged RanQL or RanTN, and scrambled control or Rab23-siRNA7.2-treated cells; **P<0.01). Results are shown as a box plot, as described in the Materials and Methods section. (C) Bar chart tabulating the number of Kif17-positive cilia and localization of Kif17 at the distal ciliary tips (pairwise comparisons made between vector control and cells overexpressing mCherry-tagged RanQL or RanTN, and scrambled control or Rab23 siRNA7.2-treated cells). (D) Anti- transportin (Impβ2) antibodies, used for co-immunoprecipitation (IP), were incubated with NIH3T3 cell lysate co-expressing Kif17–mCitrine and Rab23QL or RanQL mutants. Kif17–mCitrine and importin β2 levels were detected by immunoblotting (IB) with anti-GFP and anti-transportin antibodies. 20 μg of total lysate was each loaded into input lanes 1 and 3. (E) Proposed involvement of Rab23 in the ciliary transport of Kif17. In the cytosol, GTP–Rab23 interacts and enhances binding of Kif17 to importin β2 (Imp β2). Formation of this tripartite complex could be essential for targeting of Kif17 to the base of the primary cilium. The ciliary– cytosolic Ran-GTP–Ran-GDP gradient maintained across the ciliary barrier subsequently guides the complex into the primary cilium. Within the cilium compartment, GTP-Ran binds to importin β2, resulting in a dissociation of Kif17 and Rab23. The released Kif17 is now transported along axonemal microtubules to accumulate at the distal ciliary tip. Journal of Cell Science

3004 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 2996-3008 doi:10.1242/jcs.163964 indicated that Rab23 is mainly affiliated with ciliary membranes and NIH3T3 and hTERT-RPE1 cells (Fig. 5; supplementary material cilioplasm. This is in accordance with how the Rab-GTP–Rab-GDP Fig. S2H). Rab23 likely facilitates binding of Kif17 to importin β2, molecular switch is associated with Rab proteins alternating because the interaction between importin β2 and Kif17 was mostly between being attached to membrane and being soluble, cytosolic lost upon Rab23 depletion (Fig. 5C). Conceivably, Rab23-mediated proteins (Pylypenko and Goud, 2012; Seabra and Wasmeier, 2004). regulation of Kif17 ciliary import could be through promoting the interaction between Kif17 and its transport carrier, importin β2. Rab23 has a role in the ciliary trafficking of Kif17 Formation of this ternary complex could in turn be important for We have observed that manipulation of Rab23 levels did not targeting of Kif17 to the primary cilium. Our GST–Rab23 pulldown significantly abrogate cilia formation, suggesting that Rab23 does assays further suggest that Kif17 in the lysate binds Rab23 in vitro, not play a crucial role in the building of the ciliary architecture. in a manner that might even be stoichiometrically independent of However, enrichment of overexpressed Rab23 at the primary cilia importin β2. However, as we were unable to detect direct physical could still point to a ciliary function. We discovered that ciliary association between recombinant GST–Rab23 and His6–Kif17 localization of Kif17 depended on the GTP-binding status of Rab23 alone in the absence of cell lysate, Rab23–Kif17 interaction in vivo (and thus its presence at the primary cilium) as well as Rab23 appears to require other cellular factors. expression levels in both NIH3T3 and hTERT-RPE1 cells. An increase in the ciliary localization of Kif17 following non- Rab23 and Ran work independently to regulate ciliary degradable Rab23 expression in Rab23-shRNA-treated cells transport of Kif17 further indicated that loss of ciliary Kif17 was specifically due to Increased loss of the Kif17 ciliary localization observed after Rab23 insufficient levels of Rab23 (Fig. 3). Notably, in assessing the depletion and constitutive active RanQL overexpression, as well as ciliary localization of Kif17, Rab23 silencing in NIH3T3 and the differing effect of overexpression of Ran and Rab23 on the hTERT-RPE1 cells led to significant reductions in the ciliary interaction between importin β2 and Kif17, all imply that these intensities of Kif17 and the number of Kif17-positive cilia; small GTPases likely have separate roles in mediating ciliary however, the number of Kif17-positive cilia exhibiting transport of Kif17 (Fig. 6A–D). Although Rab23 is first required for localization of Kif17 at the distal ends were not significantly binding of Kif17 to importin β2, influx of Kif17 across the ciliary different, suggesting that Rab23 was likely to be more crucial in barrier could be subsequently determined by the ciliary–cytosolic targeting of Kif17 towards the primary cilium than in its transport Ran-GTP–Ran-GDP gradient. Perhaps similar to the process of from the ciliary base to the distal tip. Taken together with ciliary nuclear import into the nucleoplasm (Azuma and Dasso, 2000; FRAP experiments that showed Rab23-knockdown cells exhibiting Madrid and Weis, 2006), upon Kif17 import into the cilium, Ran– almost no recovery of ciliary Kif17 over time compared to GTP would associate with importin β2 and thus release Kif17 and scrambled control cells, there is strong evidence that Rab23 could Rab23 into the ciliary compartment (Fig. 6E). be required for the trafficking of cytoplasmic (unbleached) Kif17 As mentioned, Rab GTPases are known modulators of multiple towards the primary cilium. stages of exocytic and endocytic membrane trafficking in eukaryotic cells. In cycling between GDP-bound and GTP-bound states, Rab Different ciliary cargoes likely require specific regulators for proteins are recruited to specific membrane compartments, where their ciliary transport they come in close proximity and interact with distinct effector The influence of Rab23 over Kif17 ciliary trafficking prompted us proteins. Rab–effector complex formation then allows for the to question the specificity of the effects observed. Analysis of execution of precise intracellular trafficking steps, for instance, another Rab protein, Rab8a, which is essential in cilium formation vesicle motility. Since the 1990s, numerous reports describe that and ciliary transport (Follit et al., 2010; Moritz et al., 2001; Nachury Rab proteins are involved with the regulation of the microtubule et al., 2007; Omori et al., 2008), could help indicate whether Kif17 network (Horgan and McCaffrey, 2011). For example, Echard and ciliary targeting could be relying on general or specific ciliary colleagues have reported a direct, GTP-dependent interaction trafficking regulators. In our investigations, neither overexpression between Rab6A and Kif20A (kinesin-6 family) (Echard et al., of a dominant-negative Rab8a mutant or Rab8a-specific silencing 1998). A more recent paper has demonstrated how the mammalian resulted in any apparent perturbations in ciliary localization of Cos2 homologue Kif7 (kinesin-4 family), which is essential in Kif17 (supplementary material Fig. S3A–D). Co- relaying the signal transduction from smoothened to the Gli family immunoprecipitation with anti-Rab8a antibodies further showed of transcription factors, is more than likely required in formation of no detectable interaction between Rab8a and Kif17–mCitrine or the ciliary structure than transport of hedgehog signaling ciliary importin β2 (supplementary material Fig. S3F), suggesting that proteins (He et al., 2014). Kif7, like Kif17, localizes to the distal Rab8a is not likely to be crucial for the ciliary trafficking of Kif17. ciliary tips or microtubular plus ends, but it is unclear whether By contrast, although Rab8a bound to fibrocystin-CTS and Rab23 could have a role in its ciliary transport. influenced its ciliary localization, Rab23 did neither. The To date, Kif17 has been found to be involved in the regulation of influence of Rab23 on ciliary localization of Kif17 is therefore NMDA receptor subunit 2B (GluN2B)-containing vesicles in relatively specific (supplementary material Fig. S4E,J). cultured hippocampal dendrites and ciliary targeting of cyclic- nucleotide-gated (CNG) channels to the non-motile cilia of Rab23 exists in a complex with Kif17 and importin β2 olfactory sensory neurons (Guillaud et al., 2003; Jenkins et al., Control of the ciliary entry of Kif17 is known to crucially depend on 2006; Setou, 2000; Yin et al., 2012). Based on the strong association two components: the nuclear and ciliary import transport carrier of Rab23 with the primary cilium, its high levels of expression in importin β2, and the ciliary–cytoplasmic Ran-GTP–Ran-GDP neurons (Guo et al., 2006), and the involvement of Rab23 in Kif17 gradient (Dishinger et al., 2010). Our co-immunoprecipitation ciliary trafficking, Rab23 could be required in the transport of the studies showed Rab23 to be an additional player in ciliary transport known cargoes of the motor protein, which are, together, essential of Kif17. Rab23 exists in a complex with Kif17 and importin β2, for various neuronal functions. Beyond the neuronal context, the and this interaction is enhanced in a GTP-dependent manner in both role of the ciliary transport of Rab23 could also extend across Journal of Cell Science

3005 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 2996-3008 doi:10.1242/jcs.163964 various cell types, such as fibroblasts and epithelial cells. The Plasmid DNA transfection was performed using Lipofectamine™ current findings could hopefully contribute towards our 2000 reagent (11668-019, Invitrogen Life Technologies), whereas understanding of how Rab23-null mutations could lead to the Lipofectamine™ RNAiMAX reagent (13778-150, Invitrogen Life pleiotrophic Carpenter’s syndrome in humans (Alessandri et al., Technologies) was used in the siRNA-mediated knockdown of Rab23 and Rab8a. The 27-mer siRNA duplexes targeted against mouse Rab23 2010; Jenkins et al., 2007), and partly connect some similarities ′ ′ ’ cDNA are: siRNA2, 5 -GACCUAACAAACAAAGGACCAAGAGAA-3 ; observed between the symptoms of the Carpenter s syndrome and siRNA7, 5′-GAUGCAUGAAUCAUCCAGCAGAUCGAU-3′ (in siRNA7.2 ciliopathies. knockdown treatment, an equimolar mixture of siRNA7 and siRNA2 was prepared before silencing); and siRNA3, 5′-GCUCGUACAACCAUUGC- MATERIALS AND METHODS GUAUGUUUCU-3′. hsiRNA1, 5′-AAGAAUGAGGAAGCUGAGGCA- DNA constructs CUGGCA-3′ and hsiRNA2, 5′-AACAAGAUUGAUCUUCUGGAUGA- Full-length Rab23 was obtained by PCR and other standard procedures from UUCT-3′ were designed to target human Rab23. The sequence that targets a mouse brain cDNA library (Guo et al., 2006). Single point mutations giving mouse Rab8a is based on 5′-GAAUAAGUGUGAUGUGAAUGACAA- rise to Rab23 Q68L, S23N and T150A mutants were generated using the GAG-3′. The Rab23-specific shRNA sequence 5′-GGACATACTTTACA- GeneTailor™ site-directed mutagenesis kit (12397-014, Invitrogen Life GAAAG-3′, kindly provided by Dr Heidi Liou and Dr Eyleen Goh (Duke- Technologies, Carlsbad, CA). Rab23WT and mutants were subcloned into NUS Graduate Medical School, Singapore), targets the 5′ untranslated either pCIneo (E1841, Promega, Madison, WI, USA) or pmCherry-C1 region (UTR) of mouse Rab23. The sequence was cloned with or without vectors (632524, Clontech Laboratories Inc., Mountain View, CA, USA). the GFP reporter into the retroviral pUEG vector (a modification of the Wild-type Rab23 was also cloned into a pGEX-4T-1 expression vector (28- pSIREN-RetroQ-ZsGreen1 vector) (Goh et al., 2008). Retroviruses 9545-49, GE Healthcare Life Sciences, Buckinghamshire, UK) and containing scrambled and Rab23 shRNA were generated by calcium transformed into BL21 (DE3) Escherichia coli cells (C6000-03, Invitrogen phosphate transient transfection of the retroviral plasmids into HEK293GP Life Technologies) for production of glutathione-S-transferase (GST)–Rab23 cells. Freshly collected 0.45-μm-filtered retroviral supernatant was then used fusion proteins. Both pmCherry-RanQ69L (human; 30309) and pTK21-Ran to infect recipient NIH3T3 cells. T24N (human; 37396) were purchased from Addgene (Addgene, Cambridge, MA). These constructs were separately contributed by the Jay Brenman Co-immunoprecipitation, affinity pulldown assays and western (Kazgan et al., 2010) and Iain Cheeseman (Kiyomitsu and Cheeseman, 2012) blot analyses laboratories. To ensure uniformity in the comparison of Ran mutants, the Ran Cultured cells were harvested in lysis buffer (50 mM Tris-HCl pH 7.4, T24N fragment was isolated from pTK21 and inserted into the 150 mM NaCl, 1% Triton X-100, 1% NP-40), containing fresh additions of – pmCherry-C1 vector. Kif17 mCitrine [pmCit-N1 (Dishinger et al., 2010)] protease inhibitor cocktail (11873580001, Roche Diagnostics Corporation) was obtained from Kristen J. Verhey (University of Michigan Medical and 0.1 mM phenylmethanesulfonylfluoride (PMSF; P7626, Sigma- School, Ann Arbor, Michigan, MI). For ciliary FRAP analysis, Kif17 was Aldrich). For GTPγs and GDP treatments (consistently carried out prior subcloned in frame with C-terminal mCherry reporter [pmCherry-N1 to binding), cell lysate was loaded with 40 μM of GTPγs (20-176, EMD (632523, Clontech Laboratories Inc.)]. Gregory J. Pazour (University of Millipore Corporation) or GDP (20-177, EMD Millipore Corporation). In Massachusetts Medical School, Worcester, MA) and Wolfgang E. Kühn the immuno-depletion of importin β2 from cell lysate before affinity (University Medical Centre, University of Freiburg, Germany) provided the pulldown, 1 µg of anti-transportin [or mouse control IgG (sc-2025, Santa – GFP fibrocystin-CTS [pJAF99 (Follit et al., 2010)] and pLXSN- Cruz Biotechnology) in mock-depleted lysate] and protein-G–Sepharose SmoothenedA1-Venus [SmoothenedA1 is a constitutive active mutant that beads (17-0618-02; GE Healthcare Life Sciences) were added to 1 mg of consists of a mutation of the 549 tryptophan residue to leucine residue total cell lysate, and left to incubate at 4°C for 2 h. After centrifugation, (Boehlke et al., 2010)] constructs, respectively. Wild-type Arl13b tagged with supernatant was carefully collected for subsequent steps. ™ a C-terminal GFP reporter was acquired from an OriGene TrueORF cDNA For co-immunoprecipitation experiments, cell lysate, relevant antibodies clone library (MG206808, OriGene Technologies, Inc., Rockville, MD). [or rabbit control IgG (sc-2027, Santa Cruz Biotechnology)] and Sepharose affinity beads [protein-A–Sepharose beads (17-0974-01, GE Healthcare Antibodies Life Sciences) or cyanogen bromide (CNBr)-activated Sepharose 4B beads Rabbit antibodies against Rab23 were generated by repeated immunization (17-0430-01, GE Healthcare Life Sciences)] were allowed to bind with recombinant His6-tagged Rab23 fusion proteins (Guo et al., 2006). overnight. For affinity pulldown assays, GST-tagged proteins bound to Commercial antibodies include: anti-acetylated α-tubulin (6-11B-1 clone, glutathione–Sepharose-4B beads (GE Healthcare) were incubated with cell T6793, Sigma-Aldrich, St Louis, MO), anti-Arl13b (17711-1-AP, Proteintech lysate and allowed to bind overnight. After thorough washing to ensure Group Inc., Chicago, IL), anti-pericentrin (ab4448, Abcam, Hong Kong), maximal removal of non-specific binding, bead-bound ligands were eluted anti-GFP (ab5450, Abcam), anti-Kif17 (ab11261, Abcam), anti-transportin with SDS sample buffer [with the addition of 100 mM dithiothreitol (DTT)]. (558660, BD Pharmingen Inc., San Diego, CA), anti-β-actin (AC-74 clone, Denatured proteins were separated by SDS-PAGE and electro-blotted onto A2228, Sigma-Aldrich) and anti-Rab8 (610844, BD Biosciences, San Jose, nitrocellulose membranes. Membranes were incubated with primary CA) antibodies. Fluorescein isothiocyanate (FITC)-, Texas Red (TxR)- or antibodies, such as anti-Rab23 (1:2500), anti-β-actin (1:5000), anti-GFP Cy5-conjugated secondary antibodies used in immunofluorescence assays (1:1000), anti-Kif17 (1:500) and anti-transportin (1:1000) antibodies, and were purchased from Jackson ImmunoResearch Laboratories, Inc., West their respective HRP-conjugated secondary antibodies (1:5000). Grove, PA, and horseradish peroxidase (HRP)-conjugated secondary Chemiluminescent protein bands were captured using the Chemidoc™ antibodies used in western immunoblotting were purchased from Thermo MP Imaging System (Bio-Rad Laboratories Inc.). Alternatively, Fisher Scientific Inc., Waltham, MA. polyacrylamide gels were stained with Coomassie Brilliant Blue R-250 solution (161-0400, Bio-Rad Laboratories Inc.) for the visualization of GST Cell culture and transfection fusion proteins. ImageJ 1.45 software was used in the quantification of NIH3T3 fibroblasts were grown in Dulbecco’s modified Eagle’s medium western blot band intensities. Densitometric data were all presented as bar (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium charts created in Microsoft Excel. pyruvate (11360-070, Gibco Life Technologies, Grand Island, NY) and 1× penicillin-streptomycin (15140-122, Gibco Life Technologies). hTERT- Immunofluorescence assays and confocal microscopy imaging RPE1 epithelial cells were maintained in 1:1 mixture of DMEM and Ham’s Cells were fixed with 3.7% paraformaldehyde (P6148, Sigma-Aldrich) and F-12 medium supplemented with 5% FBS and 1× penicillin-streptomycin. permeabilized using 0.05% saponin (S7900, Sigma-Aldrich) for 15 min at All cells were grown at 37°C with 5% CO2 levels. To induce cilium room temperature. Primary (1:200) and secondary (1:400) antibodies, formation, cells were incubated with low-serum medium (basal medium prepared in blocking buffer [5% FBS and 2% bovine serum albumin (BSA) containing 0.5% FBS) for 24–48 h. diluted in 1× PBS], were directly incubated with the coverslips. Antibodies Journal of Cell Science

3006 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 2996-3008 doi:10.1242/jcs.163964 against acetylated α-tubulin and Arl13b were typically used for the marking Author contributions of primary cilia, whereas anti-pericentrin antibody was used to stain the Y.S.L. and B.L.T. designed the experiments and wrote the manuscript. Y.S.L. pericentriolar matrix surrounding the basal bodies. Hoechst 33342 (H1399, performed the experiments and statistical analyses. Molecular Probes Inc, Eugene, OR), prepared at a 1:10,000 dilution, was used as a nuclear counterstain. Funding Immunofluorescence images were captured with the Carl Zeiss LSM710, This work is supported by a National University of Singapore Graduate School for Integrative Sciences and Engineering (NGS) Incentive Grant; and a National LSM700 (Carl Zeiss, Oberkochen, Germany) (Core facilities, Department University Health System Bridging Fund. of Biochemistry, National University of Singapore, Singapore) or Olympus FV1000 (Confocal Microscopy Unit, Yong Loo Lin School of Medicine, Supplementary material National University of Singapore, Singapore) confocal imaging systems, Supplementary material available online at and presented as collapsed z-stacks in full resolution. ZEN 2012 edition http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.163964/-/DC1 (Carl Zeiss), FV10-ASW 4.0 Viewer (Olympus), Imaris 6.1.5 and Adobe Photoshop CS6 software were used for editing and analysis of images. References Image montages were usually displayed in x-y view, unless otherwise stated. Alessandri, J.-L., Dagoneau, N., Laville, J.-M., Baruteau, J., Hébert, J.-C. and For a clearer depiction of the localization at Rab23 at the primary cilia, z- Cormier-Daire, V. (2010). RAB23 mutation in a large family from Comoros Islands Am. J. Med. Genet. A views – a z-plane projection of stacked z-slices – were also included. In with . 152A, 982-986. Azuma, Y. and Dasso, M. (2000). The role of Ran in nuclear function. Curr. Opin. particular, the ciliary intensities of ciliary cargoes were measured using the Cell Biol. ‘ ’ 12, 302-307. histogram function in the ZEN 2012 software. The final, normalized Boehlke, C., Bashkurov, M., Buescher, A., Krick, T., John, A.-K., Nitschke, R., ciliary intensity value was calculated by subtracting the cell background Walz, G. and Kuehn, E. W. (2010). Differential role of Rab proteins in ciliary intensity from the mean fluorescence intensity. All localization data for trafficking: Rab23 regulates smoothened levels. J. Cell Sci. 123, 1460-1467. ciliary cargoes was consolidated from at least three independent experiments Bond, A. E., Dudley, E., Tuytten, R., Lemiere,̀ F., Smith, C. J., Esmans, E. L. and and presented as bar charts, created in Microsoft Excel. For the Newton, R. P. (2007). Mass spectrometric identification of Rab23 ′ ′ quantification of the intensities of ciliary cargoes, bar charts were plotted phosphorylation as a response to challenge by cytidine 3 ,5 -cyclic monophosphate in mouse brain. Rapid Commun. Mass Spectrom. 21, so that the top (white box) and bottom (grey box) lines of each column mark 2685-2692. the 75th percentile and 25th percentile of the data set respectively. The Cantagrel, V., Silhavy, J. L., Bielas, S. L., Swistun, D., Marsh, S. E., Bertrand, center line marks the median and standard errors span the mean (‘diamond’). J. Y., Audollent, S., Attié-Bitach, T., Holden, K. R., Dobyns, W. B. et al. (2008). The numbers of cells sampled for each condition are included near the Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome. bottom of all bar charts. Am. J. Hum. Genet. 83, 170-179. Chia, W. J. and Tang, B. L. (2009). Emerging roles for Rab family GTPases in human cancer. Bioch. Biophys. Acta 1795, 110-116. Ciliary fluorescence recovery after photobleaching Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and NIH3T3 cells were left to adhere onto 35-mm glass-bottomed dishes Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Nature overnight (81151, iBidi GmbH, Martinsried, Planegg, Germany). At ∼18– Sonic hedgehog gene function. 383, 407-413. Denning, K. M., Smyth, P. C., Cahill, S. F., Finn, S. P., Conlon, E., Li, J., Flavin, 24 h before imaging, cells were maintained in serum-free DMEM without R. J., Aherne, S. T., Guenther, S. M., Ferlinz, A. et al. (2007). A molecular Phenol Red dye (21063-029, Gibco Life Technologies). To minimize focus expression signature distinguishing follicular lesions in thyroid carcinoma using shifts, a 37°C temperature and 5% CO2 level were stably maintained within preamplification RT-PCR in archival samples. Mod. Pathol. 20, 1095-1102. an enclosed chamber. Using the Carl Zeiss LSM710 confocal microscope, Dishinger, J. F., Kee, H. L., Jenkins, P. M., Fan, S., Hurd, T. W., Hammond, J. W., the ciliary localization of Kif17 at the distal tips was photobleached using Truong, Y. N.-T., Margolis, B., Martens, J. R. and Verhey, K. J. (2010). Ciliary 100% laser power at 500 iterations. Images were then captured at 1-min entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP. Nat. Cell Biol. intervals over 20 min at a lower 30% laser power. For each image, six 12, 703-710. z z Echard, A., Jollivet, F., Martinez, O., Lacapere, J.-J., Rousselet, A., Janoueix- additional -slices were taken above and below the -slice of the focus (the Lerosey, I. and Goud, B. (1998). Interaction of a Golgi-associated kinesin-like total number of z-slices taken per image was 13). Net fluorescence intensity, protein with Rab6. Science 279, 580-585. after normalizing the total intensity of Kif17 against the corresponding Eggenschwiler, J. T. and Anderson, K. V. (2000). Dorsal and lateral fates in the background staining (within the same z-slice), was then plotted in a phase- mouse neural tube require the cell-autonomous activity of the open brain gene. decay graph using GraphPad Prism version 6.00 for Windows, GraphPad Dev. Biol. 227, 648-660. Software (La Jolla, CA). Eggenschwiler, J. T., Espinoza, E. and Anderson, K. V. (2001). Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature 412, 194-198. 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Rab23, a negative regulator of hedgehog signaling, localizes to the pairwise comparison. Otherwise, for non-normal or a non-parametric data plasma membrane and the endocytic pathway. Traffic 4, 869-884. distribution, a Wilcoxon rank-sum test was applied to each pairwise Follit, J. A., Li, L., Vucica, Y. and Pazour, G. J. (2010). The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J. Cell Biol. 188, 21-28. comparison. In all bar charts, error bars represent s.e.m. Gherman, A., Davis, E. E. and Katsanis, N. (2006). The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Acknowledgements Nat. Genet. 38, 961-962. We sincerely thank Dr Kristen J. Verhey for Kif17–mCitrine; Dr Gregory J. Pazour for Goh, E. L. K., Young, J. K., Kuwako, K., Tessier-Lavigne, M., He, Z., Griffin, J. W. GFP–fibrocystin-CTS; Prof. Wolfgang E. Kühn for SmoothenedA1-Venus plasmids; and Ming, G.-L. 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The authors declare no competing or financial interests. patterns in the developing spinal cord. Development 120, 3119-3130. Journal of Cell Science

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